DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 01/16/2026 has been entered.
Priority
This application claims benefit of provisional application 63/321,432 filed 03/18/2022. Applicant’s claim for the benefit of a prior-filed application under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, 365(c), or 386(c) is acknowledged.
Status of the Claims
Claims 1 and 21 were amended. Claims 7-9 and 22-24 were cancelled. Claim 25 is new.
Claims 1, 3-6, 10, 15-21 and 25 are pending (claim set filed 01/16/20259) and are examined on the merits herein.
Withdrawal of Rejections
The response and amendment filed on 01/16/20256 are acknowledged. All of the amendment and arguments have been thoroughly reviewed and considered.
For the purposes of clarity of the record, the reasons for the Examiner's withdrawal and/or maintaining if applicable, of the substantive or essential claim rejections are detailed directly below and/or in the Examiner's response to arguments section.
New Rejections
Claim Rejections - 35 USC § 112
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claim 25 is rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
Claim 25 is directed to step (b) of claim 21, i.e. recovering acetaldehyde from the product mixture as it is being produced. Claim 25 recites concentrations of the substrates of the reaction (ethanol and furfuryl), products of the reaction (acetaldehyde and furfuryl alcohol), biocatalyst, biocatalyst cofactor and buffer, time and temperature of the reaction and parameters of gas stripping. Claim 25 recites transferring at least 56% of the acetaldehyde from the product mixture to the gas. Example 2 of the specification (paragraph 0035) describes transferring of 56% of acetaldehyde from the product mixture to the gas wherein the product mixture is formed of 0.15 M ethanol, 0.032 M acetaldehyde, 0.012 M furfural, 0.037 M furfuryl alcohol and 0.1 M sodium phosphate buffer that corresponds to the concentrations recited in claim 25. Parameters of gas stripping, including time of 180 min and temperature of 22° C, gas flow rate of 240 cubic centimeters per min and pressure of 1 atm correspond to the limitations in claim 25. However, Example 2 does not recite presence of the biocatalyst and biocatalyst cofactors and hence reaction of acetaldehyde production is not being catalyzed and recovery of 56% of acetaldehyde occurs at constant amount of acetaldehyde and not as it is being produced as required by claim 21, claim 25 depends upon. Example 1 of the specification describes the reaction with the recited amount of biocatalyst, 0.2 mg/ml and the biocatalyst cofactor, NAD+, 0.5 mM (paragraphs 0029, 0030), however, Example 1 does not describe recovery of acetaldehyde.
Therefore, the recitation of transferring 56% of acetaldehyde under the recited conditions is not supported by the specification. Thus, since the support for the claim 25 is not provided, claim 25 contains new matter. One of ordinary skill in the art would not conclude that the applicant would have been in possession of the subject matter of claim 25 at the time of filing application.
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 1, 3-6, 10, 15-20 and 25 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 1 recites “furfural to cap the free amine groups” is step (a) and “additional furfural” in step (c) exposed “to the immobilized biocatalyst of step (b) … under conditions that … reduce the additional furfural to furfuryl alcohol to provide a product mixture comprising … unreacted additional furfural”. Additional furfural is not defined in the specification and is interpreted as furfural added on step (c). It is not clear how only the additional furfural is reduced to provide the unreacted additional furfural and furfural used to cap the free amine groups is not reduced since for complete capping of the free amine groups furfural is expected to be added in access. The scope and boundaries of claim 1 are not certain making claim 1 indefinite.
Claims 3-6, 10 and 15-20, dependent on claim 1, do not resolve the issue mentioned above and are rejected.
Claim 25 recites the limitation "the reaction mixture comprises the ethanol at 150 mM,
the furfural at 12 mM, the biocatalyst at 0.2 mg/ml, the biocatalyst cofactor at 0.5 mM, acetaldehyde at 32 mM, furfuryl alcohol at 37 mM and sodium phosphate buffer at 100 mM". Claim 21, claim 25 depends upon, recites “a reaction mixture comprising the biocatalyst, the biocatalyst cofactor, the ethanol and the furfural”. A reaction mixture of claim 21 does not recite comprising acetaldehyde and furfuryl alcohol, which are the products of reactions. It is not clear if Applicant refers to a reaction mixture of claim 21 in claim 25 or intended to describe composition of the product mixture in claim 25 rather than the reaction mixture since claim 21 recites the same components in the product mixture (the acetaldehyde, the furfuryl alcohol, the biocatalyst, the biocatalyst cofactor, unoxidized ethanol, and unreduced furfural) and the specification (paragraph 0035) describes the same components and at the same concentrations in the product mixture comprising co-reactants and co-products. The scope and boundaries of claim 25 are not certain making claim 1 indefinite.
Claim 25 is interpreted as directed to the product mixture comprising the recited components.
Maintained/Modified Rejections
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1, 3-6, 10 and 15-20 are rejected under 35 U.S.C. 103 as being unpatentable over Li (Li et al. Biodegradation, 2011, 22, 1227-1237 on record in IDS) in view of Leskovac (Leskovac et al. FEMS Yeast Research, 2002, 481-494 on record in IDS), Marques (Marques et al. Applied Surface Science, 2013, 275, 347-360) and Clarizia (Clarizia et al. 2020, 393, 123425, 1-7) as evidenced by Shinde (Shinde et al. Biotechn. Reports, 2018, e00260, 1-7), Bootorabi (Bootorabi et al. BMC Biochemistry, 2008, 9:32, 1-8) and Sachdeva (Sachdeva et al. Green Process Synth., 2012, 1, 469-477).
Regarding claims 1, Li teaches ethanol-dependent reduction of furfural by alcohol dehydrogenases (Title). Li discloses that yeast alcohol dehydrogenases, specifically YADH1, YADH6, and YADH7 (which are isozymes), are shown to reduce furfural with biocatalyst cofactors NADH for YADH1 and NADPH for YADH6, and YADH7 as the reducing power (p. 1228, left column, 2nd paragraph). Li describes that the coupled reaction for ethanol oxidation to acetaldehyde and furfural reduction to furfuryl alcohol, shown in Equations 4-6 (p. 1234, right column), provides a thermodynamically more favorable method of producing acetaldehyde from ethanol. Li discloses kinetic parameters for reaction of acetaldehyde production from ethanol with four alcohol dehydrogenases, one of which is YADH1 (p. 1233, Table 1). Further, Li teaches that YADH1 is constitutively expressed and its kinetic parameters for NADH-dependent reduction of furfural suggest that it can effectively catalyze furfural reduction at high furfural concentrations (p. 1236, left column, 2nd paragraph). Therefore, Li teaches a method of producing acetaldehyde by exposing ethanol and furfural to a biocatalyst (YADH1) and a biocatalyst cofactor (NADH) dispersed in the reaction mixture to produce a product mixture comprising furfuryl alcohol and acetaldehyde. It would have been obvious to one of ordinary skill in the art that the product mixture will also contain unoxidized ethanol, unreduced furfural and biocatalyst cofactor.
Li does not teach yeast YADH2 and YADH3 as biocatalyst producing acetaldehyde and does not teach immobilization of the biocatalyst on a solid support. Although Li teaches concentration of acetaldehyde from the after reaction termination by HPLC (p. 1229, right column, 2nd paragraph), Li does not teach recovering of the acetaldehyde from the reaction mixture as it is being produced with oxygen free gas.
Leskovac teaches structure, function and mechanism of action of the three yeast alcohol dehydrogenase isoenzymes, YADH-1, YADH-2 and YADH-3 (Abstract). Leskovac describes that yeast alcohol dehydrogenases catalyze the reversible reaction of conversion of ethanol to acetaldehyde with NAD+ as a cofactor and determines kinetic parameters for that reaction for three isoenzymes (p. 482, left column 1st paragraph and Table 1). Leskovac discloses that YADH-1 and YADH-3 have very similar kinetic characteristics, while YADH-2 has a much higher substrate specificity for ethanol and acetaldehyde, and much lower Michaelis constants with ethanol and acetaldehyde. It was found that for all alcohols, normalized rates with YADH-2 were about three-fold faster than with YADH-1 (p. 482, left column, 1st paragraph).
Marques teaches enzyme immobilization and chemical functionalization of surfaces (Abstract). Marques discloses the use of amine-functionalized solid supports of modified polystyrene and glutaraldehyde (GA) as a bi-functional cross linking agent for enzyme immobilization. The enzyme layer is covalently bonded to the amine group of the solid polystyrene support using the linker, glutaraldehyde (p. 350-351, section 2.4 and 2. 5). Marques describes that GA acts as a bifunctional linker, forming stable covalent bonds between the chemical groups from the support with amine group of the enzyme (p. 353, right column, 2nd paragraph) and mentions that the immobilized enzymes have retained their enzymatic activities (p. 355, left column, 3rd paragraph). Therefore, Marques teaches the claimed solid support for enzyme immobilization. Yeast alcohol dehydrogenase can be immobilized by its amine group with the linker GA as evidenced by Shinde (Abstract). Shinde mentions that immobilization increases YADH thermostability and reusability (Abstract).
Marques describes that glutaraldehyde is removed by washing prior to binding the enzyme (p. 350, section 2.4). It can be envisaged that the solid support may have exposed amine groups not involved in the interaction with glutaraldehyde since the unreacted amine groups were not blocked prior to enzyme binding. The unreacted amine groups can interact with produced acetaldehyde via Schiff base as evidenced by Bootorabi (Abstract) and hence need to be blocked or capped. The method of acetaldehyde production based on Li involves furfural as a substrate which has carbonyl group that can interact with amine group with formation of Schiff base as evidenced by Sachdeva. Sachdeva teaches that furfural and its derivatives efficiently bind to the amine group of alanine when stirring in aqueous solution at room temperature (p. 471, right column, 2nd paragraph, p. 474, left column, 1st paragraph, Table 1, 3g - furfural). Therefore, furfural can be used to cap the unreacted amine groups of the surface to prepare solid support for immobilization of YADH isozymes. It would have been obvious to one of ordinary skill in the art that additional amount of furfural can be added for the reaction of acetaldehyde production to replenish furfural used for capping the free amine groups.
Clarizia teaches efficient recovery and purification of acetaldehyde during ethanol oxidation by removing acetaldehyde from the solution carried out by injecting an inert gas (nitrogen) into the solution and collecting the gas by feeding the resulting gaseous stream to an absorber, in which acetaldehyde is captured by the aqueous stream (Abstract, p. 6, right column, 6th paragraph). Clarizia discloses that almost all acetaldehyde produced by ethanol oxidation (93%) was separated from the aqueous mixture after using inert gas stripping (p. 5, left column, 3rd paragraph).
First, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the coupled reaction method of Li to include isozymes YADH2 and YADH3 taught by Leskovac and try to use three isoenzymes together. One would have been motivated to do so since all three isoenzymes were shown by Leskovac to produce acetaldehyde, Leskovac teaches that YADH3 has similar kinetic characteristics as YADH1 and since it is expected to perform similarly in the coupled reaction method of Li and YADH2 has a much higher substrate specificity for ethanol and acetaldehyde, and much lower Michaelis constants with ethanol and acetaldehyde and can increase efficiency of acetaldehyde production in coupled method of Li. A skilled artisan would have reasonably expected success in the combination because Li and Leskovac teach yeast alcohol dehydrogenases producing acetaldehyde.
Second, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to use the immobilization method of Marques in the coupled reaction method of acetaldehyde production based on Li and Leskovac teachings for immobilization of biocatalyst. One would have been motivated to do so since Marques showed that GA acts as a bi-functional linker, forming stable covalent bonds between the chemical groups from the support with amine group of the enzyme and enzyme retains activity. YADH can be immobilized by its amine group via GA linker resulting in the increase in its thermostability and reusability as evidenced by Shinde. A skilled artisan would have reasonably expected success in the combination because Li and Leskovac provide method of acetaldehyde production from ethanol with YADH catalyzing that reaction, Shinde describes immobilization of YADH via GA linker and Marques provides method of enzyme immobilization on solid surface via GA linking.
Third, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention that furfural used as a substrate during acetaldehyde production based on Li and Leskovac teachings can block the unreacted amino groups of the solid support prior to enzyme immobilization taught by Marques. One would have been motivated to suggest that since unreacted amine groups can react with the product of reaction acetaldehyde as evidenced by Bootorabi, furfural can interact with amine group with formation of Schiff base in aqueous solution at room temperature as evidenced by Sachdeva and application of the same compound as the substrate of the reaction will avoid possible interference of additional substances present in the reaction mixture and not involved in the reaction. It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to add additional amount of furfural for the reaction. One would have motivated to do that with reasonably expected success since furfural used for amino groups capping cannot participate in YADH reaction and it is within the skill of the artisan in the field to determine the amount of the substrate necessary for catalytic reaction.
Last, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to add the recovery of acetaldehyde described by Clarizia to the coupled reaction method based on Li, Leskovac and Marques teachings. One would have been motivated to do so since Clarizia teaches that almost all (93%) acetaldehyde produced was separated from the aqueous mixture after using inert gas stripping. A skilled artisan would have reasonably expected success in the combination because Li, Leskovac and Clarizia teach production of acetaldehyde from ethanol.
Thus, combination of teachings of Li, Leskovac, Marques and Clarizia as evidenced by Shinde, Bootorabi and Sachdeva renders claim 1 obvious.
Regarding claim 10, Li teaches that lignocellulosic biomasses can be converted into sugars and sugar derivatives, such as furfural, wherein the sugars may be fermented into ethanol (Abstract). Thus, teachings of Li, Leskovac, Marques and Clarizia as evidenced by Shinde, Bootorabi and Sachdeva render claim 10 obvious.
Regarding claims 3-6, Clarizia teaches the use of an inert gas containing nitrogen (p. 4, left column, last paragraph).Clarizia discloses that the nitrogen stream is employed at 0.8 L/min and at a pressure of 1 atm (p, 4, right column, 1st paragraph) that reads on claimed limitations.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to follow guidance of Clarizia and add step of recovery of acetaldehyde by injecting inert gas, nitrogen, at the specified parameters of flow rate and pressure to the coupled reaction method of acetaldehyde production based on Li, Leskovac and Marques teachings. One would have been motivated to do so since Clarizia teaches that almost all (93%) acetaldehyde produced was separated from the aqueous mixture after using inert gas stripping. A skilled artisan would have reasonably expected success in the combination because Li, Leskovac and Clarizia teach production of acetaldehyde from ethanol. Thus, teachings of Li, Leskovac, Marques and Clarizia as evidenced by Shinde, Bootorabi and Sachdeva render claims 3-6 obvious.
Regarding claims 15-19, the combination of prior art of Li, Leskovac, Marques and Clarizia teaches the claimed method for production of acetaldehyde from ethanol and renders claim 1 obvious as described above. Li teaches a method of producing acetaldehyde by exposing ethanol and furfural to a biocatalyst (YADH1) and a biocatalyst cofactor (NADH) to produce a product mixture comprising furfuryl alcohol and acetaldehyde. Leskovac describes three isozymes of YADH, YADH1, YADH2 and YADH3, from which YADH3 has the same kinetic parameters as YADH1 from Li teaching and YADH2 has a much higher substrate specificity for ethanol and acetaldehyde, much lower Michaelis constants with ethanol and acetaldehyde and is about three-fold faster than YADH1, providing motivation to include all three isoenzymes of YADH in the method for producing acetaldehyde of Li to increase its efficiency. Marques teaches method of immobilization of enzymes of the solid surface via glutaraldehyde bifunctional linker providing retention of enzymatic activity and YADH can be immobilized by its amine group via GA linker resulting in the increase in its thermostability and reusability as evidenced by Shinde and that provides motivation to immobilize YADH on the solid surface as described by Marques. Clarizia teaches efficient method of acetaldehyde recovery as it being produced with almost all (93%) acetaldehyde produced separated from the aqueous mixture after using inert gas stripping providing motivation to include the described recovery step to Li method of acetaldehyde production. Thus, combination of Li, Leskovac, Marques and Clarizia teaches all the claimed limitations of the method of acetaldehyde production from ethanol of claim 1. Therefore, one of ordinary skill in the art would expect that the method of acetaldehyde production taught by Li, Leskovac, Marques and Clarizia would necessarily yield the claimed conversion for ethanol and furfural and selectivity for acetaldehyde and furfuryl alcohol. Thus, teachings of Li, Leskovac, Marques and Clarizia as evidenced by Shinde, Bootorabi and Sachdeva render claims 15-19 obvious.
Regarding claim 20, Leskovac teaches structure, function and mechanism of action of the three yeast alcohol dehydrogenase isoenzymes, YADH-1, YADH-2 and YADH-3 (Abstract) and discloses that YADH-1 and YADH-3 have very similar kinetic characteristics, while YADH-2 has a much higher substrate specificity for ethanol and acetaldehyde, and much lower Michaelis constants with ethanol and acetaldehyde. It was found that for all alcohols, normalized rates with YADH-2 were about three-fold faster than with YADH-1 (p. 482, left column, 1st paragraph).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to add isozymes YADH2 and YADH3 taught by Leskovac to the catalytic reaction of acetaldehyde production with YADH1 taught by Li and try to use three isoenzymes together. One would have been motivated to do so with reasonable expectation of success since all three isoenzymes were shown by Leskovac to produce acetaldehyde, Leskovac teaches that YADH3 has similar kinetic characteristics as YADH1 and since it is expected to perform similarly in the coupled reaction method of Li and YADH2 has a much higher substrate specificity for ethanol and acetaldehyde, and much lower Michaelis constants with ethanol and acetaldehyde and hence can increase efficiency of acetaldehyde production in coupled method of Li. Thus, teachings of Li, Leskovac, Marques and Clarizia as evidenced by Shinde, Bootorabi and Sachdeva render claim 20 obvious.
Claims 21 and 25 are rejected under 35 U.S.C. 103 as being unpatentable over Li (Li et al. Biodegradation, 2011, 22, 1227-1237 on record in IDS) in view of Leskovac (Leskovac et al. FEMS Yeast Research, 2002, 481-494 on record in IDS) and Clarizia (Clarizia et al. 2020, 393, 123425, 1-7).
Regarding claims 21, Li teaches ethanol-dependent reduction of furfural by alcohol dehydrogenases (Title). Li discloses that yeast alcohol dehydrogenases, specifically YADH1, YADH6, and YADH7 (which are isozymes), are shown to reduce furfural with biocatalyst cofactors NADH for YADH1 and NADPH for YADH6, and YADH7 as the reducing power (p. 1228, left column, 2nd paragraph). Li describes that the coupled reaction for ethanol oxidation to acetaldehyde and furfural reduction to furfuryl alcohol, shown in Equations 4-6 (p. 1234, right column), provides a thermodynamically more favorable method of producing acetaldehyde from ethanol. Li discloses kinetic parameters for reaction of acetaldehyde production from ethanol with four alcohol dehydrogenases, one of which is YADH1 (p. 1233, Table 1). Further, Li teaches that YADH1 is constitutively expressed and its kinetic parameters for NADH-dependent reduction of furfural suggest that it can effectively catalyze furfural reduction at high furfural concentrations (p. 1236, left column, 2nd paragraph). Li describes reaction to comprise biocatalysts not immobilized but dispersed in the reaction mixture (p. 1229, right column, 2nd paragraph). Therefore, Li teaches a method of producing acetaldehyde by exposing ethanol and furfural to a biocatalyst (YADH1) and a biocatalyst cofactor (NADH) dispersed in the reaction mixture to produce a product mixture comprising furfuryl alcohol and acetaldehyde. It would have been obvious to one of ordinary skill in the art that the product mixture will also contain unoxidized ethanol, unreduced furfural and biocatalyst cofactor.
Li does not teach yeast YADH2 and YADH3 as biocatalyst producing acetaldehyde. Although Li teaches concentration of acetaldehyde from the after reaction termination by HPLC (p. 1229, right column, 2nd paragraph), Li does not teach recovering of the acetaldehyde from the reaction mixture as it is being produced with oxygen free gas.
Leskovac teaches structure, function and mechanism of action of the three yeast alcohol dehydrogenase isoenzymes, YADH-1, YADH-2 and YADH-3 (Abstract). Leskovac describes that yeast alcohol dehydrogenases catalyze the reversible reaction of conversion of ethanol to acetaldehyde with NAD+ as a cofactor and determines kinetic parameters for that reaction for three isoenzymes (p. 482, left column 1st paragraph and Table 1). Leskovac discloses that YADH-1 and YADH-3 have very similar kinetic characteristics, while YADH-2 has a much higher substrate specificity for ethanol and acetaldehyde, and much lower Michaelis constants with ethanol and acetaldehyde. It was found that for all alcohols, normalized rates with YADH-2 were about three-fold faster than with YADH-1 (p. 482, left column, 1st paragraph).
Clarizia teaches efficient recovery and purification of acetaldehyde during ethanol oxidation by removing acetaldehyde from the solution carried out by injecting an inert gas (nitrogen) into the solution and collecting the gas by feeding the resulting gaseous stream to an absorber, in which acetaldehyde is captured by the aqueous stream (Abstract, p. 6, right column, 6th paragraph). Clarizia discloses that almost all acetaldehyde produced by ethanol oxidation (93%) was separated from the aqueous mixture after using inert gas stripping (p. 5, left column, 3rd paragraph).
First, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the coupled reaction method of Li to include isozymes YADH2 and YADH3 taught by Leskovac and try to use three isoenzymes together. One would have been motivated to do so since all three isoenzymes were shown by Leskovac to produce acetaldehyde, Leskovac teaches that YADH3 has similar kinetic characteristics as YADH1 and since it is expected to perform similarly in the coupled reaction method of Li and YADH2 has a much higher substrate specificity for ethanol and acetaldehyde, and much lower Michaelis constants with ethanol and acetaldehyde and can increase efficiency of acetaldehyde production in coupled method of Li. A skilled artisan would have reasonably expected success in the combination because Li and Leskovac teach yeast alcohol dehydrogenases producing acetaldehyde.
Second, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to add the recovery of acetaldehyde described by Clarizia to the coupled reaction method based on Li and Leskovac teachings. One would have been motivated to do so since Clarizia teaches that almost all (93%) acetaldehyde produced was separated from the aqueous mixture after using inert gas stripping. A skilled artisan would have reasonably expected success in the combination because Li, Leskovac and Clarizia teach production of acetaldehyde from ethanol.
Thus, combination of teachings of Li, Leskovac and Clarizia renders claim 21 obvious.
Regarding claim 25, Clarizia teaches that almost all acetaldehyde produced by ethanol oxidation (93%) was separated from the aqueous mixture after using inert gas stripping for 120 min (p. 5, left column, 3rd paragraph). The initial amount of acetaldehyde was 24 mM. The parameters of stripping were: the nitrogen stream is employed at 0.8 L/min and at a pressure of 1 atm at 25°C (p. 5, left column, 2nd paragraph). Li teaches that 170 mM ethanol and 17 mM furfural can reach equilibrium of 154 mM ethanol, 1.1 mM furfural, 16 mM furfuryl alcohol and 16 mM acetaldehyde (p. 1234, left column, 2nd paragraph). Li mentions using 0.13 mg/ml and 0.16 mg/ml of two alcohol dehydrogenases, 10 µM NAD+ and 50 mM KPi buffer (p. 1230, left column, last paragraph, right column, 1st paragraph). Parameters of gas stripping in Clarizia teaching, i.e. concentration of acetaldehyde, flow rate and pressure of nitrogen gas and temperature and time of stripping are the same or close to instant parameters. Concentration of co-reactants and co-products described in equilibrium scenario of Li are close to instant concentrations, except concentration of NAD+ and furfural. However, since claim is directed to percent of acetaldehyde transferred to the gas and not the concentration of acetaldehyde produced in the reaction, does not actually require the enzymatic reaction to proceed, parameters of acetaldehyde gas stripping (acetaldehyde concentration, time of stripping, flow rate and pressure of the gas) are given more weight rather than concentrations of the components of product mixture. Clarizia teaches 24 mM acetaldehyde that is close to instant 32 mM, 120 min stripping at 25°C, that is close to instant 180 min at 22°C and 1 atm gas pressure that is the same as instant pressure. Clarizia discloses 800 cm3/min flow rate versus instant 240 cm3/min. However, it is noted that "where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation" (See MPEP 2144.05 II).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to expect that gas stripping of acetaldehyde produced by the recited product mixture and method based on Li and Leskovac teachings and by applying parameters of stripping as taught by Clarizia will transfer at least 56% of acetaldehyde to gas. One would have been motivated to do so since Clarizia teaches that almost all (93%) acetaldehyde produced was separated from the aqueous mixture after using inert gas stripping applied to similar to instant acetaldehyde concentration and with similar parameters. A skilled artisan would have reasonably expected success in the combination because Li, Leskovac and Clarizia teach production of acetaldehyde from ethanol.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention that parameters of acetaldehyde recovery by gas stripping based on Clarizia teaching, including gas flow rate, temperature and time of stripping, can be optimized. One would have been motivated to do so to increase recovery of acetaldehyde. A skilled artisan would have reasonably expected success in the optimization because selection of reaction parameters is routine and conventional.
Thus, combination of teachings of Li, Leskovac and Clarizia renders claim 25 obvious.
Response to Arguments
Applicant's arguments filed 01/16/2026 have been fully considered but they are not persuasive.
Regarding independent claim 1, Applicant argues (addressing p. 6 of the Remarks) that amended claim 1 “requires furfural capping per steps (a)-(b) prior to the exposure step (c), a step which also requires the use of additional furfural. None of the cited prior art teaches or leads the person of ordinary skill to each and every element of independent claim 1, including steps (a)-(b) carried out prior to the exposure step (c), a step also requiring the use of additional furfural”. These arguments are not persuasive because:
Marques teaches enzyme immobilization on polystyrene support via glutaraldehyde (p. 350-351, section 2.4 and 2. 5), however, does not teach blocking the unreacted amino groups of the support. Since the unreacted amine groups can interact with produced acetaldehyde via Schiff base as evidenced by Bootorabi (Abstract) that provides motivation to block or cap unreacted amino groups. Furfural can react with amino group and thus block it as evidenced by Sachdeva (Table 1, 3g - furfural) and since furfural is one of the substrates of the reaction of production of acetaldehyde from ethanol in Li teaching (Fig. 3) that motivates to use furfural for blocking and not another substance that can interfere with the reaction. When furfural is used for capping it forms Schiff base and cannot be used as a substrate thus motivating to add additional amount of furfural for the reaction.
Regarding independent claim 21, Applicant argues (addressing p. 6-7 of the Remarks) that claim 21 requires enzymes to be not immobilized but dispersed in the reaction mixture and “the person of ordinary skill have no reason to adopt Clarizia's gas-stripping process since the significant disadvantage of enzyme denaturation would have been expected to preclude the intended purpose of the Li/Leskovac method”. Applicant further argues that the results of method of claim 21 are unexpected and demonstrated only via the disclosure of present application and its co-inventors. Applicant notes that new claim 25 recites conditions to achieve the recited transfer of at least 56% of acetaldehyde and refers to Examples 1 and 2 and previously submitted Declaration. Applicant argues that results of Clarizia teaching are irrelevant since Clarizia method is entirely different and concludes that: “Absent impermissible hindsight in view of the present application, there is no reasonable expectation of success for such a method.” These arguments are not persuasive because:
In response to applicant's argument that the examiner's conclusion of obviousness is based upon improper hindsight reasoning, it must be recognized that any judgment on obviousness is in a sense necessarily a reconstruction based upon hindsight reasoning. But so long as it takes into account only knowledge which was within the level of ordinary skill at the time the claimed invention was made, and does not include knowledge gleaned only from the applicant's disclosure, such a reconstruction is proper. See In re McLaughlin, 443 F.2d 1392, 170 USPQ 209 (CCPA 1971). In instant case, although Clarizia teaches different non-enzymatic method of acetaldehyde production, the high efficiency of the method of acetaldehyde recovery described by Clarizia provides motivation to use the gas stripping method with expectation of recovery of acetaldehyde produced based on Li and Leskovac teachings.
The advantage of recovering acetaldehyde from the reaction mixture as it is being produced recognized by the Applicant does not make the combination of prior art non-obvious because the prior art does not need to point out all advantages if there is a motivation to combine the prior art. MPEP 2145: “The fact that appellant has recognized another advantage which would flow naturally from following the suggestion of the prior art cannot be the basis for patentability when the differences would otherwise be obvious." Ex parte Obiaya, 227 USPQ 58, 60 (Bd. Pat. App. & Inter. 1985). In instant case, Li, Leskovac and Clarizia teach production of acetaldehyde from ethanol and Clarizia discloses method allowing to separate almost all (93%) acetaldehyde produced from the aqueous mixture after using inert gas (p. 5, left column, 3rd paragraph) providing motivation to add acetaldehyde recovery to method of Li and Leskovac.
Regarding the unexpected results of claims 21 and 25 reciting transfer of at least 56% of acetaldehyde as it is being produced, these results are not supported by the specification and claim 25 represents new matter as described in 112(a) rejection. The Declaration Applicant refers to describes collection of produced acetaldehyde in the nitrogen stream, however does not provide the percent of acetaldehyde recovery. Example 1 describes the catalytic reaction of acetaldehyde and furfuryl alcohol production and analysis of the products by gas chromatography, but does not include acetaldehyde recovery in nitrogen stream. Example 2 describes recovery of 56% acetaldehyde from the product mixture formed by combining ethanol, furfuryl, acetaldehyde and furfuryl alcohol in sodium phosphate buffer having concentrations recited in claim 25, however, the product mixture of Example 2 does not have biocatalyst and biocatalyst cofactor and hence there is no catalytic reaction. Therefore, Example 2 does not provide evidence to recovery of at least 56% of acetaldehyde as it is being produced. Additionally, claim 25 does not require the enzymatic reaction to proceed. Recovery of acetaldehyde from the product mixture in the absence of enzymatic reaction and hence absence of possible denaturation of protein biocatalyst due to air-water interface is similar to method of Clarizia teaching disclosing 93% recovery from 24 mM acetaldehyde during 120 min of stripping with nitrogen (p. 5, left column, 2nd paragraph). Therefore, the recovery of at least 56% of acetaldehyde based on combination of teachings of Li, Leskovac and Clarizia is expected.
Thus, the 35 U.S.C. 103 rejection is maintained and modified necessitated by amendment of claims.
Conclusion
No claims are allowed.
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/L.G.K./Examiner, Art Unit 1653
/SHARMILA G LANDAU/Supervisory Patent Examiner, Art Unit 1653
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